3.6. Supernatant Analysis for Cell Death Through Pyroptosis: LDH Release Assay Kit

Thaw frozen supernatants at room temperature as described in Subheading 3.5, step 1.

Thaw LDH control well, collect media, and spin in a microcentrifuge tube for 20 min at 20,000 × g (4 °C). Have a no-cell control, containing only fresh MGM 10/10 as a negative control.

Set up a 96-well flat bottom plate and add 50 μL aliquots from controls and samples. Follow kit protocol. In this case, a microplate reader that analyzes 490 or 492 nm is necessary.

Calculate the results according to kit protocol and analyze data through graph-generating software, as explained in Subheading 3.5, step 4. In this case, the graph is comparing LDH release percentage in the different samples.

For biological relevance, collect data from three independent experiments, and analyze and compare them through a statistical test (one-way ANOVA, two-way ANOVA, Mann-Whitney, etc.), according to the experiment goals.